Purpose: to evaluate the seroprevalence of infection caused by HSV-2 among pregnant women delivering at the University Hospital, Faculty of Medicine of Ribeirão Preto (UHFMRP-USP) and to standardize laboratory techniques to be used for this purpose. Methods: a total of 1500 blood samples from pregnant women seen at the Obstetric Center of the Department of Gynecology and Obstetrics, UHFMRP-USP, between January 1st and October 31st, 1996, were evaluated. To determine the real prevalence of HSV-2 infection, the ELISA technique was standardized but, during its initial use, it was found to be not sufficiently specific to discriminate between the two viral types (75%). Thus, it became necessary to use a more specific technique and the method standardized for this purpose was Western blot, which can detect the specific HSV-2 viral protein. Results: the seroprevalence of herpes infection induced by the two viral types (HSV-1 and HSV-2) was 94.5% when ELISA was used. With the use of Western blot, a 32% seroprevalence of HSV-2 infection was detected in the studied population, whether symptomatic or asymptomatic. Conclusion: a high prevalence of carrier status for HSV-2 and HSV-1 infection was detected, as shown by the high rate of positivity for antibodies against this virus. ELISA did not show sufficient specificity to discriminate between HSV-2 and HSV-1 antibodies.